Biology Reference
In-Depth Information
supernatant removed. With tissue, they must be pretreated
with proteinase K to breakdown the interstitial structure to
dislodge the cells which are centrifuged (1,200 rpm, 3 min)
yielding the cell pellet.
3. The cell pellet is washed with PBS twice at room temperature
before resuspending in PBS to a concentration of 5×10 5
cells/ml.
4. Suffi cient paraformaldehyde is added to a fi nal concentration
of 1% and incubated at room temperature for 10 min. This
cross-linking reaction is terminated by adding 1 M glycine to a
fi nal concentration of 0.1 M.
5. The cross-linked cells are pellet (1,200 rpm, 3 min) and washed
once more with ice-cold PBS.
6. Then, it is resuspended in 6 ml of Lysis Buffer and mixed
gently before incubating on ice for 10 min.
7. At this stage, the outer membrane of the cells is solubilized and
a further centrifugation (2,000 rpm, 5 min) step will yield the
crude nuclear extract as the pellet.
8. The crude nuclear extract is washed once with ice-old PBS
before resuspending in 1.9 ml of High Salt Lysis Buffer.
9. Afterward, this crude nuclear extract is subjected to multiple
short pulses of sonication on ice to shear the chromosome yield-
ing short fragments of 500-1,000 bases. The magnitude and
the number of pulses are dependent on the type of sonicator
used which should be optimized before the start of this assay.
10. Then the extract is further centrifuged (10,000 rpm, 15 min,
4°C) and the supernatant would contain the chromosome
fragments with and without bound protein together with other
nuclear proteins.
11. The protein concentration of this supernatant is determined,
and 100-500 mg of protein should be used for the subsequent
immunoprecipitation step.
12. For the immunoprecipitation step, the protein/DNA mixture
can be precleared by adding protein-A-sepharose beads and
incubate for 30 min at 4°C. This preclearing step can minimize
the background due to nonspecifi c binding of DNA/proteins
onto protein-A-sepharose beads slurry.
13. Add the primary antibody recognizing the protein of interest
and incubate overnight at 4°C.
14. Then, add 50 ml of protein-A-sepharose slurry and incubate for
another 2 h at 4°C.
15. Harvest the protein-A-sepharose beads linked to the antibody-
protein-DNA complex by centrifugations at 12,000 rpm for
30 s and place it on ice.
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